The pharmaceutical industry has wider applications of human liver hepatocytes. These cells enable the study of drug metabolism, drug-drug interactions, and drug toxicity. The assays on primary hepatocyte cells considerably reduce the time for drug development by facilitating high-throughput screening of numerous drug leads simultaneously. The initial screening aids in the selection of suitable leads for further studies, thus lowering expense, number of animal studies, time, and labour.
However, the primary human hepatocyte culture remains challenging, despite its vast applications. It hinders the in vitro studies, especially for the assessment of hepatotoxicity required for regulatory approvals. Therefore, over the years, several approaches have emerged to address the problems with hepatocyte culture. This blog explores the limitations of culturing Primary Human Hepatocytes and the techniques to resolve them.
Limitations of Primary Human Hepatocyte Culture
Primary human hepatocytes are the gold standard for measuring drug metabolism and hepatic research. However, their in vitro culture faces quite a few hurdles that obstruct research on them.
Scarcity of Cells: Human liver hepatocytes are typically isolated from patients undergoing liver surgery, which offers a resected tissue sample. Additionally, whole tissue is also available from healthy livers that were unfit for transplant. The advancing technologies, however, have substantially reduced the need for surgical interventions, leading to low availability of tissue for cell isolation.
Cell Yield: The amount of viable cells that can be isolated from a tissue depends on the isolation procedure. Good tissue quality and faster isolation are recommended for higher cell viability. It requires proper transportation of tissue to the laboratory and expertise for increasing the final purity and yield of hepatocytes.
Limited Proliferation: Primary cells generally have short in vitro proliferative capacity. However, the proliferation of primary hepatocyte cells declines as they tend to differentiate in the two-dimensional conditions, losing their physiological functions. The differentiation begins occurring in a week. It necessitates setting assays on hepatocytes within a few passages. The deviation from their metabolic properties also impedes long-term studies.
Strategies to Culture Hepatocytes
Numerous applications of Human Liver Hepatocytes have prompted scientists to search for ways to extend the retention of their in vivo properties in the in vitro conditions. Here are a few approaches.
Medium Composition: The medium for hepatocytes is either William E or DMEM. Different supplements have been added to the medium to improve the metabolic capacity. For instance, hepatocyte growth factor, vitamin C, and oncostatin M improved cell metabolism. Addition of insulin, glucocorticoids (such as dexamethasone or hydrocortisone), and growth factors (like EGF) also sustains hepatic functions in primary hepatocyte cells. Depending on the study requirements, medium with or without serum can be used.
Extracellular Matrix (ECM): Cell attachment to the substrate is essential for their survival and proliferation. Primary human hepatocyte cultures usually employ collagen coating on flasks or dishes for cell adherence and expansion. Coating with Matrigel (laminin-rich collagen gel) is also frequently used. Additionally, optimal cell density is considered a crucial aspect of cell attachment and survival.
Cell Lines: To extend the in vitro lifespan of hepatocytes, immortalization of human hepatocytes has also been a vital tool. Transfection with viral oncogenes or hTERT overexpression has been the popular immortalization techniques. A few examples of human hepatocyte cell lines are HepG2, HepaRG, Huh-7, PLC/PRF/5, etc. However, they exhibit low resemblance to hepatic behavior, which restricts their use to limited studies.
Sandwich Culture: This culture allows the growth of a monolayer of hepatocytes on a matrix, followed by covering the monolayer with another matrix to form another layer of hepatocytes. It results in the culturing of cells in a sheet similar to that in the tissue. It extends the proliferative capacity of these cells but can also restrict their application in specific assays due to their sheet-like growth. The type of matrix under and above the cells affects the cell behavior and therefore, the study results.
Co-culture: Interaction of hepatocytes with other liver cells is essential to sustain hepatocyte survival and function within the tissue. Culture of primary human hepatocytes with non-parenchymal cells mimic these interactions. For example, hepatocyte co-culture with endothelial cells or fibroblasts promotes their functions. Co-culture models with stellate cells or Kupffer cells provide knowledge about tissue pathophysiology.
Spheroid: Imitating the in vivo environment of the tissue has propelled the 3D culture techniques with spheroids and organoids at their forefront. Hepatocyte spheroids have gained attention due to their close resemblance to tissue behavior and prolonged survival in culture.
Additionally, many other strategies, such as fluid-flow systems, optimizing oxygen tension, gene editing, etc., can overcome the limitations of 2D hepatocyte culture.
Conclusion
Hepatocytes are a vital component of drug development. However, even today its culture faces several challenges. Over the past decades, scientists have pursued different directions to optimize the culture conditions and sustain the hepatic function for a longer time period. With the rising amount of research on these cells and the lower availability of human tissue samples, these strategies have become more necessary. Kosheeka delivers primary human hepatocytes and also provides guidance on their culture by expert scientists to assist in your research endeavors.
